This proposal is led by Novasite, with the concourse of Axon Instruments and the Univ. of New Mexico (UNM). We propose a novel HTS system for Drug Discovery capable of screening 10 3 variant targets simultaneously in real time, at little additional costs relative to one-target screening systems. We will use this instrumentation to develop a novel approach to Drug Discovery via large scale generation and screening of variant targets, centered on identifying ligand-receptor interactions at large scale. The advantage is a combinatorial explosion in the number of ligand- receptor interactions explored relative to one-receptor screening approaches (from 10 3 to 10 8, for 10 3 libraries). Our aim is the discovery of an anti-inflammatory and immunosuppressive agent, by discovering an agonist for the cannabinoid-2 G protein coupled receptor (GPCR). We have set up a general expression system that efficiently tranfects one single variant target cDNA per cell in a massive transfection step. We use a cell-based GPCR functional assay based on Ca2+ sensitive fluorescence dyes. In the first Specific Aim, Axon will develop a novel fluorescence microscope imaging system capable of visualizing (screening) thousands of individual cells (variant targets) simultaneously in one well at High Throughput. This system brings HTS to a new level, where each cell is a functional assay by itself, the physical limit for a cell-based assay. We randomize the receptor's binding site residues, resulting in thousands of receptor constructs with enhanced recognition properties, capable of recognizing novel and high affinity leads. For lead optimization, the UNM component aims at developing a special HTS flow cytometry system to sort cells with significantly lower EC5Os than the wild type receptor. The variant GPCR present in these isolated cells, identified by PCR, represents a mutation that enhances the potency of a given lead. This data will be analyzed by computational molecular models, attempting to match the variation of chemical moieties within the lead compound with the variation of amino acid residues within the receptor to guide docking procedures. Bioinformatic analysis of these biochemically-derived computational models will be used to translate the identified amino acid changes that enhance the lead's potency into mirror-image modifications proposed on the chemical compound that will guide the lead optimization process. In year 1 we will set up the individual components mentioned above; year 2 Axon delivers a prototype HTS imaging system (Lead Identification); year 3 UNM delivers adapted flow cytometer (lead optimization): years 4-5 develop drug lead and reach HTS.